Pear pomace alleviated atopic dermatitis in NC/Nga mice and inhibited LPS-induced inflammation in RAW 264.7 macrophages

BACKGROUND/OBJECTIVES Poorly regulated inflammation is believed to be the most predominant factor that can result in a wide scope of diseases including atopic dermatitis (AD). Despite many studies on the effect of pear pomace in obesity-related disorders including dysregulated gut microbiota, the protective effect of pear pomace in AD is still unknown. This study aimed to evaluate the effect of pear pomace ethanol extract (PPE) on AD by inhibiting inflammation. MATERIALS/METHODS In the in vivo experiment, 2, 4-dinitrochlorobenzene (DNCB) was applied to NC/Nga mice to induce AD-like skin lesions. After the induction, PPE was administered daily by oral gavage for 4 weeks. The clinical severity score, serum IgE levels, spleen weight, histological changes in dorsal skin, and inflammation-related proteins were measured. In the cell study, RAW 264.7 cells were pretreated with PPE before stimulation with lipopolysaccharide (LPS). Nitrite oxide (NO) production and nuclear factor kappa B (NF-kB) protein expression were detected. RESULTS Compared to the AD control (AD-C) group, IgE levels were dramatically decreased via PPE treatment. PPE significantly reduced scratching behavior, improved skin symptoms, and decreased ear thickness compared to the AD-C group. In addition, PPE inhibited the DNCB-induced expression of inducible nitrite oxide synthase (iNOS), the receptor for advanced glycation end products, extracellular signal-regulated kinase (ERK) 1/2, and NF-κB. PPE inhibited the LPS-induced overproduction of NO and the enhanced expression of iNOS and cyclooxygenase-2. Moreover, the phosphorylation of ERK1/2 and NF-κB in RAW 264.7 cells was suppressed by PPE. CONCLUSIONS These results suggest that PPE could be explored as a therapeutic agent to prevent AD.

INTRODUCTION
Atopic dermatitis (AD) is a chronic, relapsing, and highly itching inflammatory skin disease [1,2]. In Europe and the Unites States, approximately 20% of children and 7% to 14% of adults NC/Nga mice have been used as an AD model for human dermatitis since 1957 by Kondo et al. [10]. NC/Nga mice will become dry and scaly skin under conventional conditions [10,11]. Skin lesions also developed immunologically and histologically around neck and back within several weeks. At the same time, serum IgE level moderately increased demonstrating a biochemical change [10,12]. Elevated IgE levels are the key to the pathogenesis of AD, resulting in the activation of mast cells and leading to the release of mediators such as histamine [13][14][15][16]. These mediators were positively correlated with skin lesions like prurience and eczema [14][15][16]. Thus, controlling the inflammation status in AD is thought to be important.
Pears, one of the oldest plants cultivated by humans, are consumed all over the world [17]. Yet, pear pomace, the main waste byproduct in pear processing, is hitherto poorly understood. Some studies have suggested that pear pomace showed beneficial effects in increasing fecal bulking, toxic component binding, and the regulation of gut microbiota structures in highfat diet-fed rats due to the high dietary fiber content [18][19][20][21]. In our preliminary results, pear pomace water extract (PPW) inhibited adipocyte differentiation by inducing apoptosis in the early stages of differentiation and reduced hepatic lipid peroxidation [22,23]. In addition, pear pomace ethanol extract (PPE) effectively reduced insulin resistance in vitro and in vivo [24]. We also found that PPE was more effective at inhibiting inflammation than PPW in RAW 264.7 cells after lipopolysaccharide (LPS) stimulation (data not shown). However, the anti-inflammatory effect of PPE, especially in AD, has not been examined.
In the present study, we hypothesized that PPE could prevent AD by regulating inflammatory responses in 2, 4-dinitrochlorobenzene (DNCB)-induced AD in NC/Nga mice and LPSinduced inflammation in RAW 264.7 macrophage. In addition, we measured whether nuclear factor kappa B (NF-κB) was involved in the action mechanism of PPE.

Preparation of PPE
The ethanol extract of pear pomace was prepared according to previous protocols [24]. Briefly, pear pomace, the cake left after extracting the juice, was extracted with 50% ethanol, filtered, freeze-dried, and stored in a −80°C freezer. The yield obtained was 27.5%

Induction of AD using DNCB
DNCB was an efficient tool to induce AD in NC/Nga mice within a short time period topical application. We slightly modified the AD protocol according to published papers [25][26][27]. After 2 week of acclimatization, the dorsal hair of NC/Nga mice was removed using an electric shaver (hair clipper model 6000AD; Thrive Japan, Kawasaki, Japan) and depilatory cream (Veet; Reckitt, Slough, UK). To induce AD-like skin lesions, the mice were divided into 2 groups (normal control [Cont] group and AD group); DNCB (Sigma-Aldrich, St. Louis, MO, USA) was applied to the AD group. The mice were sensitized with 200 uL of 1% DNCB solution (dissolved in a 3:1 mixture of acetone and olive oil) twice over a week. Then, the dorsal skin was challenged with 150 uL of 0.4% DNCB solution 3 times per week for 7 weeks. Three weeks after the challenge, the AD group was further divided into 3 groups: AD control (AD-C), AD + PPE 200 (PPE 200 mg/kg body weight), and AD + PPE 400 (PPE 400 mg/kg body weight). PPE extract was dissolved in distilled water and administered daily by oral gavage for 4 weeks. Distilled water was administered to the Cont and AD-C groups. Fig. 1A illustrates the schematic diagram of AD induction and treatment with PPE.

Measurement of basic characteristics in NC/Nga mice
To investigate the scratching behavior of DNCB-induced AD in NC/Nga mice, the mice were placed individually in clear cages. Seven weeks after sensitization, the mice were treated with PPE upon 0.4% DNCB treatment. For 20 min after DNCB treatment, the total number of mice scratching themselves only with the hind paw was counted; the scores ranged from 0 to 4: 0 (no scratching), 2 (scratching for less than 1.5 sec), and 4 (scratching for more than 1.5 sec). The clinical severity score for dermatitis was evaluated macroscopically on the day of the necropsy using a specific scoring procedure. The skin clinical severity score was expressed as the sum of the individual scores for the 4 symptoms of erythema/hemorrhage, dryness/scaling, edema, and excoriation/erosion. For each skin symptom, 0 was defined as no symptoms, 1 as mild symptom, 2 as moderate symptoms, and 3 as severe symptoms [25,28]. To determine the ear thickness, both the left and right ears of the mice were removed, and the thickness was measured using a digital caliper (Mitutoyo Corporation, Kawasaki, Japan), and calculated as the mean thickness value of both sides. Additionally, the spleen was dissected for weighing.

Measurement of serum IgE levels in NC/Nga mice
Blood samples were collected, separated immediately by centrifugation, and stored at −70°C. Serum IgE levels were determined using the mouse IgE ELISA kit (Fujifilm Wako Shibayagi Corporation, Shibukawa, Japan) following the manufacturer's instructions.

Histological analysis of NC/Nga mice
To measure the epidermal inflammation and infiltration of mast cells, dorsal skin was obtained and fixed in 10% formalin. The samples were embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E) or toluidine blue (Sigma-Aldrich). The stained sections were observed under a microscope at 100× magnification.

Cell culture and viability
Murine cell line RAW 264.7 macrophages (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (Gibco-BRL, Grand Island, NY, USA) which was supplemented with a 10% fetal bovine serum (Gibco-BRL) and 1% penicillin (Gibco-BRL). The cells were maintained at 37°C in a humidified atmosphere with 5% CO 2 . At 80% of confluence, the cells were subdivided for future experiments. To examine the cytotoxicity of PPE, the cells were seeded in a 96-well plate at a density of 10 × 10 3 cells/well and were subsequently treated with either phosphate-buffed saline (PBS), 100, or 250 ug/ mL of PPE. After 24 h of incubation, 2 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) solution (Sigma-Aldrich) was added to each well and the cells were incubated further for another 4 h. The medium was then removed, and the formazan precipitate was solubilized in dimethyl sulfoxide (Sigma-Aldrich). Absorbance was measured at 540 nm using microplate reader (Multiskan EX; Thermo Fisher Scientific, Waltham, MA, USA). The relative cell viability was calculated as the percentage of the control cells.

Measurement of nitrite oxide (NO) production in RAW 246.7 macrophages
The nitrite concentration in the culture medium was measured as an indicator of NO production. RAW 264.7 macrophages were seeded at a density of 20 × 10 4 cells/well in a 24-well plate and pretreated with PBS or PPE for 1 h before stimulation with 1 ug/mL of LPS (Sigma-Aldrich). After 24 h incubation, the culture supernatant was collected and centrifuged at 2,400 g for 10 min. The cell culture medium was combined with an equal volume of Griess reagent (Sigma-Aldrich) in a 96-well plate and incubated for 15 min at room temperature. Then, the absorbance was measured at 540 nm using microplate reader.

Western blot analysis
Mouse skin samples and cells were homogenized in ice-cold radioimmune precipitation assay buffer (Thermo Fisher Scientific) with a 1% protease inhibitor cocktail and a phosphatase inhibitor. The protein concentrations were determined by the Bradford (Sigma-Aldrich) method and equal amounts of protein (40 μg each) were separated on 10% SDS-PAGE gels and transferred onto a nitrocellulose membrane (Millipore, Billerica, MA, USA). The membrane was blocked for 1 h at 4°C with 5% skim milk in TBS-T buffer (10 mM

Statistical analysis
Statistical analyses were performed using the SPSS program (version SPSS 23; IBM, Chicago, IL, USA). The results are presented as the mean ± SE of 3 independent experiments. Differences between the groups were evaluated using one-way analysis of variance. Multiple comparisons were performed by Duncan's multiple range test. A P-value of < 0.05 was considered statistically significant.

Effect of PPE on the basic characteristics of NC/Nga mice
To induce AD-like skin lesions, NC/Nga mice were sensitized with a high dose of DNCB solution for 1 week. After sensitization, a low dose of DNCB was applied on the dorsal skin of the mice with or without PPE treatment (200 or 400 mg/kg body weight) (Fig. 1A). As shown in Fig. 1B, no significant differences in body weight were observed among the groups at the end of the experimental period, indicating that PPE could be used for in vivo applications safely with no visual side effects. Importantly, serum IgE levels in the AD-C group were notably higher by 76-fold (72.55 ug/mL) compared to 0.94 ug/mL in the Cont group. The high-dose PPE treatment reduced serum IgE levels to about 37.2% (45.53 ug/mL) compared to the AD-C group (Fig. 1C). Investigations were carried out to evaluate the clinical symptoms, scratching behaviors and skin clinic severity score. The application of DNCB remarkably increased scratching behavior; PPE at 200 and 400 mg/kg decreased scratching behaviors by 23.6% and 40%, respectively, versus the AD-C group (Fig. 1D). Additionally, PPE treatment markedly improved skin clinical severity score relative to the AD-C group (Fig. 1E). In previous studies by Ko et al. [29] and Shin et al. [30], NC/Nga mice treated with DNCB showed an enlarged spleen, the organ responsible for maintaining systemic immune responses. Spleen weight in the AD-C group markedly increased compared to that in the Cont group, whereas PPE treatment lowered spleen weight (Fig. 1F). Furthermore, the thickness of the skin and ear was also suppressed by PPE treatment (Fig. 1G and H).

PPE reduced the aggravation of AD-like skin lesions in DNCB-induced NC/Nga mice
To investigate the histological changes in the dorsal skin of NC/Nga mice, skin samples were stained with H&E and toluidine blue. A thicker epidermis was observed in the dorsal skin of AD-C specimens compared to specimens from the Cont group, whereas specimens from the PPE groups exhibited decreases in epidermal thickness ( Fig. 2A). The regulation of mast cells in the allergic response is an essential strategy in autoimmune diseases such as AD [31]. The AD-C group showed significantly higher numbers of mast cells in the dorsal skin than the Cont group. Nevertheless, the infiltration of mast cells diminished with PPE supplementation in AD-induced NC/Nga mice (Fig. 2B). Taken together, these results indicated that the administration of PPE attenuated AD-like lesions in DNCB-induced NC/Nga mice.

PPE inhibited inflammation markers in the skin of AD-induced NC/Nga mice
Next, we detected inflammation-related protein expression in the dorsal skin, as shown in Fig. 3. The protein levels of RAGE, a marker of inflammation, were elevated upon DNCB treatment, whereas PPE reversed those changes in the dorsal skin of AD induced NC/Nga mice. The application of DNCB drastically increased the p-ERK/ERK ratio relative to the Cont group. However, PPE dose-dependently reduced the p-ERK/ERK ratio compared to the AD-C group; moreover, PPE 400 effectively returned the p-ERK/ERK levels to those of the Cont level. In accordance with the p-ERK/ERK results, the protein expression of NF-κB showed a significant drop in the dorsal skin of the PPE groups (Fig. 3). Compared to the elevation in iNOS protein in the AD-C group, PPE treatment considerably suppressed iNOS expression (Fig. 3). Taken together, these results indicated that PPE treatment could help to prevent DNCB-induced AD in NC/Nga mice.

PPE suppressed inflammatory response in LPS-induced RAW 264.7 macrophages
Due to the anti-inflammation effect of PPE in vivo, we studied whether PPE could inhibit the inflammatory response upon LPS treatment in the RAW 264.7 macrophages. We first measured cell cytotoxicity in the RAW 264.7 cells using MTT assay. The cell viability was not different among the groups. To determine the inhibitory effect of PPE on NO production, RAW 264.7 cells were pre-treated with PPE. Stimulation with LPS remarkably aggravated NO production compared to the control; pretreatment of PPE ameliorated these increases in a dose-dependent manner (Fig. 4B). The elevated NO production in LPS treated cells was consistent with the increased expression of iNOS and COX-2 proteins (Fig. 4C)  compared to the LPS control in RAW 264.7 macrophages. Consistent with the in vivo study results, LPS significantly raised the p-ERK/ERK ratio (Fig. 4D) and PPE remarkably reduced the p-ERK/ERK ratio in LPS-stimulated cells (Fig. 4D). Furthermore, the expression of NF-κB/p65 was decreased in the PPE treated groups. These results collectively suggested that PPE could potently suppress the inflammatory response in RAW 264.7 macrophages.

DISCUSSION
In the present study, we examined whether PPE alleviated the clinical features of AD using AD-like lesions in DNCB-induced NC/Nga mice and LPS-induced inflammation in RAW 264.7 macrophages. We found that the administration of PPE inhibited AD-like lesions (Figs. 1 and 2) and suppressed inflammation-related proteins in the dorsal skin of AD-induced NC/Nga mice (Fig. 3). We also identified that PPE decreased NO production and NF-κB expression in the LPSinduced inflammatory response in RAW 264.7 cells (Fig. 4). Taken together, our work indicates that PPE may be developed as a therapeutic agent to ameliorate AD.
AD is a chronic inflammatory skin disease characterized by the hyper production of IgE. Seventy percent of AD patients showed elevated levels of IgE which was positively associated with disease severity. The patients with low IgE level slowly displayed allergic reactions due to IgE-mediated sensitization later in life [32]. allergic reaction, IgE is increased and binds to mast cells with high affinity. IgE-activated mast cells release mediators including histamine, prostaglandins, and leukotrienes. In addition, the over-secretion from mast cells results in the infiltration of inflammatory epidermal cells, macrophages, and eosinophils [15,16,28]. Recently, Emrick et al. [33] demonstrated that the Tmem79 gene, a newly identified gene in AD animals and humans, played a key role in skin inflammation and itching [34,35]. The mutation of Tmem79 in mice showed the dysregulation of inflammation, dermal thickness, and skin lesions. Interestingly, blocking mast cell degranulation decreased scratching behaviors in Tmem79 −/− mice by inhibiting histaminergic itching via the histamine receptor 1 and 4-dependent pathways [33]. These findings suggest that the reduction in mast cells may be related to the decrease in scratching behaviors. In our study, PPE remarkably reduced IgE levels and mast cell infiltration (Figs. 1C and 2B). In addition, PPE effectively inhibited scratching behavior and improved clinical symptoms ( Fig. 1D and E). Increased epidermal thickness by DNCB treatment was suppressed in the PPE-treated groups (Figs. 1G and 2A). These protective effects against the AD symptom suggest that PPE treatment may be useful for the treatment or management of AD.
RAGE is a multiligand receptor in the immunoglobulin superfamily on the cell surface and has been implicated as an amplifier of pro-inflammatory responses in inflammatory diseases [36,37]. The activation of RAGE engages key signaling pathways including ERK1/2 phosphorylation and the downstream NF-κB pathway to mediate the expression of molecules that are involved in inflammation [36]. NF-κB, composed of the p50/p65 heterodimer and the inhibitory protein IkBα, is a ubiquitous transcription factor that resides in the cytoplasm of resting cell. Once activated, phosphorylated IκBα underwent degradation and further disassociation. The remaining p50/p65 heterodimer translocate to the nucleus, where it induces the transcription of more than 400 genes of which are associated with cellular activities such as apoptosis, proliferation, metastasis, chemo resistance, and inflammation. [38,39]. Therefore, the regulation of mediators via the NF-κB pathway is a critical mechanism in the inflammatory process and a potential target for inflammation intervention. Consistent with the literature, our results indicated that PPE treatment reduced the expression of RAGE that was increased by the application of DNCB (Fig. 3). In addition, PPE treatment inhibited NF-κB expressions in the dorsal skin of NC/Nga mice (Fig. 3) and in RAW 264.7 cells (Fig. 4). Moreover, iNOS, one of the types of NOSs, is attributed to the redundant or overproduced NO during inflammation or infection [40]. Our present findings clearly displayed that iNOS expression was effectively diminished through PPE treatment both in vivo and in vitro (Figs. 3 and 4C). The downregulated iNOS expression may, in part, explain the PPE-decreased NO production in RAW 264.7 cells (Fig. 4B). Since, when NF-κB is activated, the p65/p50 heterodimer disassociates with the phosphorylation of IκBα and translocate from the cytosol to the nucleus, PPE may block the translocation of NF-κB to the nucleus and thus, down-regulate iNOS expression. However, whether PPE also interferes with the phosphorylation of IκBα remains unknown and further studies are needed.
The main active ingredients in pears are considered to be phenolic and flavonoids compounds, among which, leucocyanidin, catechin, epicatechin, chlorogenic acid, arbutin, ursolic acid, quercitrin, and quercetin account for a large proportion [41]. The unique combination of polyphenols and flavonoids may be related to the potential activity for treating allergic inflammatory diseases [33]. In addition, remarkable progress has been achieved in recent years to elucidate the anti-AD properties of quercetin [42,43]. Moreover, chlorogenic acid has been widely studied as an antioxidant in mouse RAW 264.7 macrophages [44,45] and human THP-1 macrophages [46] which can mitigate inflammatory response. However, since there has been no experimental report on the screening of the bioactive fractions and components from PPE, an arbitrary decision about which component is mainly responsible for the anti-inflammatory properties could not be made, and further research is required to clarify those issues.
In conclusion, we demonstrated that PPE was effective in decreasing AD-like lesions in DNCB-induced NC/Nga mice. The inflammatory response including NF-κB expression in the dorsal skin was inhibited by PPE treatment. PPE was also shown to ameliorate LPS-induced inflammation in RAW 264.7 cells by decreasing NO production and NF-κB expression. Taken together, PPE might represent an anti-inflammatory candidate to treat inflammatory diseases like AD.


s2fieldsofstudy:
Biology
Computer Science

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GotEnzymes: an extensive database of enzyme parameter predictions

Enzyme parameters are essential for quantitatively understanding, modelling, and engineering cells. However, experimental measurements cover only a small fraction of known enzyme-compound pairs in model organisms, much less in other organisms. Ar-tiﬁcial intelligence (AI) techniques have accelerated the pace of exploring enzyme properties by predicting these in a high-throughput manner. Here, we present GotEnzymes, an extensive database with enzyme parameter predictions by AI approaches, which is publicly available at https://metabolicatlas.org/ gotenzymes for interactive web exploration and programmatic access. The ﬁrst release of this data resource contains predicted turnover numbers of over 25.7 million enzyme-compound pairs across 8099 organisms. We believe that GotEnzymes, with the readily-predicted enzyme parameters, would bring a speed boost to biological research covering both experimental and computational ﬁelds that involve working with candidate enzymes.

INTRODUCTION
Enzymes are essential macromolecules that catalyse biochemical reactions, and thus have been interesting targets for scientific research in wide fields, e.g. biotechnology (1) and biomedicine (2). Enzyme performance can be quantitatively described by parameters such as enzyme turnover number k cat and Michaelis constant K M , which can be measured experimentally by enzyme assays, albeit in a lowthroughput manner. While past decades have witnessed an increasing number of measured parameters of enzymes for various organisms (3), the coverage of the measurements is still poor even for well-studied organisms (4,5). The coverage can be improved by a large-scale acquisition of enzyme parameters that leverages high-throughput omics data and metabolic modelling, which has been demonstrated for several model organisms (6)(7)(8)(9). However, such efforts rely heavily on organism-specific data and thus face difficulty in keeping pace with genome sequencing.
The estimation of enzyme-related parameters can be accelerated by artificial intelligence (AI) techniques based on sequence information, as exemplified by machine or deep learning-based predictions of enzyme temperature optima (10), enzyme commission (EC) number (11), turnover number (12,13) and Michaelis constant (5). Despite the successes, these methods may require users to reproduce the entire prediction pipeline in order to use the estimations it produces. Since it would be much easier to retrieve an enzyme parameter from a database rather than running the entire software stack, which incurs time and resource costs and might require expertise, we have hereby opted to create a public database containing readily-predicted enzyme parameters at a large scale, which would bring a speed boost to biological research.
To this end, we present GotEnzymes, a comprehensive database with enzyme parameter predictions freely available at https://metabolicatlas.org/gotenzymes. The database is presented in Metabolic Atlas, a platform that primarily integrates and presents open-source genome-scale metabolic models (GEMs), which have been used in systems biology for a wide range of applications (14). With GotEnzymes, modellers can begin to consider including predicted enzymatic constraints into GEMs without having to handle the case of missing values. Moreover, the GEMs provide a metabolic context in which one can place the reactions described in GotEnzymes. Thus, the implementation of GotEnzymes in the platform enriches the use of predicted enzyme parameters with the bigger picture of metabolism. Relative fraction x .

DATABASE CONTENT
The first release of GotEnzymes contains predicted turnover numbers of 25 795 560 enzyme-compound pairs, each being annotated with the EC number, across 8 099 organisms including 747 eukaryotes, 6963 bacteria and 389 archaea. As can be intuitively expected, eukaryotes generally have more turnover numbers per organism ( Figure  1A). The median turnover number for the entire dataset is 5 s −1 ( Figure 1B), and most values (75%) lie in the range between 1 and 100 s −1 , consistent with an experimental data-based study (3). By grouping the organisms, we found that the median turnover numbers for eukaryotes (4.5 s −1 ), bacteria (5.2 s −1 ) and archaea (5 s −1 ) are close although eukaryotes have the lowest median ( Figure 1B). By grouping the EC numbers, we found that while the difference is small, isomerases (EC 5.X.X.X) exhibit the highest median (7.3 s −1 ) while ligases (EC 6.X.X.X) the smallest (4 s −1 ) ( Figure 1C), which is consistent with previous findings (3).

Data collection
The input data used for the turnover number predictions were extracted from the KEGG database (15), including per organism protein sequences, compound structures, and EC number-reaction associations that link enzymes and compounds. Note that the compound structures used in the predictions are represented as molecular graphs converted from the simplified molecular-input line-entry system (SMILES), and therefore the compounds without valid defined SMILES information were excluded.

Data prediction
The pretrained deep learning-based model DLKcat (version 1.0.0) (13) was used to predict enzyme turnover numbers based on the collected protein sequences and compound SMILES information of enzyme-compound pairs. Note that the pairs with currency metabolites, e.g. water and proton, were excluded if they were not the only substrates for the enzyme. The pretrained model was downloaded from the GitHub repository: https://github.com/ SysBioChalmers/DLKcat. GotEnzymes benefits from a setup that enables it to become better with time. With a reproducible prediction pipeline and code versioning on GitHub at the repository https://github.com/feiranl/GotEnzymes, the data can be regenerated at future time points to extend predictions for other enzymes and other parameters. As can be expected from AI approaches, more and better training data can lead to improved predictions. Therefore, GotEnzymes is foreseen to receive updates in line with the improvements in training data. Moreover, with improved computational AI approaches developed in the future, the prediction modules used by GotEnzymes to predict different parameter types can be updated independently, leading to updated releases of the database.

Data implementation
For its development, GotEnzymes leverages the technical infrastructure that was built for the existing parts of the Metabolic Atlas platform. In addition to increasing the development speed, the existing platform also provides users with a richer picture of metabolism. A specific example is the use of the Cross-references section and the adjacent identifier pages, which are shared between the GEMs integrated into the platform and GotEnzymes (Figure 2). Some technical changes were required, however. To optimally handle the large tabular dataset contained in GotEnzymes, a new Postgres database was included in the software stack, next to the existing graph database Neo4j. The application programming interface (API) to this database is handled by the existing middleware, again providing development speed, and increasing maintainability.
Another aspect of the implementation is the FAIRification of GotEnzymes. The Metabolic Atlas platform has previously introduced identifiers for reactions and metabolites for the integrated GEMs. These have now been linked with Nucleic Acids Research, 2022 3 The prediction table is interactive, enabling the user to sort and apply filtering, including minimum and maximum values for the predicted turnover numbers. In the screenshot, values are filtered for the hsa organism and a minimum turnover of 1 s −1 , sorting turnover numbers decreasingly. the service Identifiers.org (16), thus facilitating their reuse by other platforms. A similar approach for FAIRifying the predictions in GotEnzymes is foreseen.

DATABASE USAGE
GotEnzymes offers interactive exploration and manual data export as tab-separated values (TSV). Moreover, to facilitate interactions with workflows and other programmatic tools, free access is also provided via the API.
Regarding interactive exploration of the website, GotEnzymes offers a fuzzy text search of EC classes, compound names, reaction names, and organism names, in addition to KEGG identifiers of the previously mentioned categories. Gene identifiers are, however, only used for exact search, in order to provide an optimum user experience with minimal impact on resources for the close to ∼5.8 million different genes in GotEnzymes.
The user can then explore the full details of the search suggestions, which contains a short table describing the selected element (Figure 2A), followed by another table for cross-references ( Figure 2B). The mapping to crossreferences that are provided by GotEnzymes is also linking to the cross-references in GEMs integrated in Metabolic Atlas ( Figure 2C). Finally, a larger table detailing the predictions applicable to the selected item is taking up the bigger part of the view ( Figure 2D). This table allows for further interaction such as sorting and filtering via text input on most columns and minimum-maximum filters for the predicted turnover numbers. Most of the identifiers listed in this table are presented as links to other pages, showing how the data is interconnected. For manual data export, the prediction table presents a button to create a TSV file of the information currently displayed in the table ( Figure 2D).
Programmatic access to the predicted enzyme parameters in GotEnzymes is available via API at https:// metabolicatlas.org/api.

DISCUSSION AND FUTURE DIRECTIONS
The BRENDA enzyme database, as the most comprehensive and widely used enzyme information resource, has collected enzyme parameters for decades (17). The number of turnover numbers in BRENDA (83 662, as reported in January 2022), which are experimentally determined, is much less than the computationally predicted numbers in GotEnzymes. With the large size of the data, we envision that GotEnzymes would bring a speed boost to biological research covering both experimental and computational fields. On one hand, GotEnzymes is ready to give the best enzymes based on the predicted parameters, which would guide enzyme selection and design, and thus reduce the time in experimental cycles such as the design-build-testlearn cycle of synthetic biology and metabolic engineering (18). On the other hand, GotEnzymes, via its API, facilitates cross-organism computational analyses, e.g. evolutionary analysis (3), and metabolic modelling dependent on large-scale enzyme parameters, e.g. kinetic models (19) and proteome-constrained models (20).
In the future, we will expand GotEnzymes by integrating more types of enzyme parameters using available AIbased predictions such as enzyme temperature optima (10) and Michaelis constant (5), thus fulfilling more aspects of users' requirements. In addition, we will implement annotations from other databases such as MetaCyc (21) and deep learning-based annotation tools (11,22) to enlarge the coverage of the enzyme-compound pairs, which were generated based only on KEGG database in the initial release. Last but not least, we intend to overlay the enzyme parameters to pathway maps on the Metabolic Atlas platform as new layers, which we foresee to enable interactive comparison and facilitate advanced model development.


A Chromosome-Scale Genome Assembly of the Okapi (Okapia johnstoni)

Abstract The okapi (Okapia johnstoni), or forest giraffe, is the only species in its genus and the only extant sister group of the giraffe within the family Giraffidae. The species is one of the remaining large vertebrates surrounded by mystery because of its elusive behavior as well as the armed conflicts in the region where it occurs, making it difficult to study. Deforestation puts the okapi under constant anthropogenic pressure, and it is currently listed as “Endangered” on the IUCN Red List. Here, we present the first annotated de novo okapi genome assembly based on PacBio continuous long reads, polished with short reads, and anchored into chromosome-scale scaffolds using Hi-C proximity ligation sequencing. The final assembly (TBG_Okapi_asm_v1) has a length of 2.39 Gbp, of which 98% are represented by 28 scaffolds > 3.9 Mbp. The contig N50 of 61 Mbp and scaffold N50 of 102 Mbp, together with a BUSCO score of 94.7%, and 23 412 annotated genes, underline the high quality of the assembly. This chromosome-scale genome assembly is a valuable resource for future conservation of the species and comparative genomic studies among the giraffids and other ruminants.

The okapi (Okapia johnstoni) ( Figure 1A, also known as forest giraffe, is a monotypic species in the genus Okapia and the only extant sister group to the giraffe (Giraffa spp.) within the family Giraffidae. Today, okapi only occurs in central, northern, and eastern regions of the Democratic Republic of the Congo and occasionally crosses the border to Uganda. The species is classified as "Endangered" on the IUCN Red List based on an estimated decline to one-half of its original population size during the last 24 years, which equals only about three generations (Mallon et al. 2015). This elusive species is difficult to observe in its natural habitat, dense rainforest, and most population estimates rely on dung-based surveys, as ongoing political conflicts, illegal mining, and poaching hinder more accurate population counts.
Genomic data for the okapi is still limited to being sequenced from non-invasive sampling strategies (e.g., dung samples) and single molecular markers, such as a few mitochondrial and nuclear genes. Analyses of these verify the presence of okapis south-west of the Congo River (Stanton et al. 2016) and characterize the genetic diversity across their geographic distribution (Stanton et al. 2014). There are two draft genome assemblies available for the okapi (Agaba et al. 2016;Dudchenko et al. 2017;Chen et al. 2019). However, both assemblies were constructed using short-read sequencing libraries only and are thus highly fragmented.
The development and increasing accessibility of long-read sequencing technologies and long-range scaffolding methods has made chromosome-scale genome assemblies for many animal and plant species possible (Bickhart et al. 2017;Marrano et al. 2020;Winter et al. 2020;Rhie et al. 2021), including the okapi's closest relative, the giraffe (Farré et al. 2019;Liu et al. 2021). The availability of these high-contiguity genomes enables complex comparative genomic studies and the understanding of the genetic factors underpinning unique evolutionary traits, such as hypertension, which is the norm in the exceptionally tall animal (Liu et al. 2021). Furthermore, highquality genome assemblies often serve as reference sequences to study species for which mapping of short-read sequences (e.g., from museum collections or feces) is the only source of genetic information.
Here, we report the first high-quality de novo genome assembly of the okapi based on PacBio continuous long reads, polished with short reads. Furthermore, we use existing chromatin interaction map (Hi-C) data (Dudchenko et al. 2017) to construct a chromosome-scale genome assembly as reference genome to study okapi genetic diversity, genome evolution, selection, and future comparative and population genomic analyses. This improved assembly of a genome from the sister group to the giraffe is an important resource to study the genetic basis and evolution of the unique characteristics of giraffids and will facilitate future genome-wide conservation genomic assessments of wild okapi populations.

Biological Materials
A kidney-tissue sample from a male okapi named Moyo (International Studbook Number 0385) was collected by veterinarians during post-mortem examination at the Zoo Antwerp and preserved at −80 °C until further processing. Figure 1. The okapi, Hi-C contact map, and Gene set completeness analyses. Painting of the okapi by Jon Baldur Hlidberg (www.fauna.is) (A). Proximity ligation contact map of the scaffolded assembly TBG_Okapi_asm_v1 (B). Gene set completeness analyses (BUSCO) using both the mammalia_odb10 (C) and the cetartiodactyla_odb10 gene sets (D) for the 3 okapi assemblies and the annotation of TBG_Okapi_asm_v1. The assembly with the highest BUSCO scores for both gene sets is TBG_Okapi_asm_v1.
High-molecular-weight genomic DNA (gDNA) was extracted using a standard phenol-chloroform extraction protocol (Sambrooks J, Russel DW 2001). DNA concentration and yield were measured using the Qubit dsDNA BR Assay Kit on the Qubit Fluorometer (Thermo Fisher Scientific) and DNA integrity was evaluated using the Genomic DNA ScreenTape on the Agilent 2200 TapeStation system (Agilent Technologies).

Nucleic Acid Library Preparation and Sequencing
A SMRTbell sequencing library was prepared using the SMRTbell Express Prep kit v2.0 Protocol (Pacific Biosciences-PacBio, Menlo Park, CA, USA) and sequenced on the PacBio Sequel II system on continuous long read (CLR) mode with the Sequel II Sequencing Kit 2.0 (PacBio). For preparing a Chromium sequencing library (10X Genomics, Inc., Pleasanton, CA, USA), the gDNA was size-selected using a BluePippin System (SageScience, Inc., Beverly, MA, USA) to remove DNA molecules < 30 kbp. The size-selected gDNA was then sent to SciLifeLab (Stockholm, Sweden) for library preparation and sequencing on the Illumina NovaSeq 6000 system.

Genome-Wide Heterozygosity
A folded Site Frequency Spectrum (SFS) was used to estimate the genome-wide heterozygosity of the two chromosomescale assemblies from the BAM files generated for PSMC. First, ANGSD v.0.933 (Korneliussen et al. 2014) (flag -doSaf 1) was used to estimate site allele frequencies. We set the minimum score for both mapping and base quality to 30, used the 95 th percentile of the sample's depth distribution as maximum depth cut-off, and enabled the extended Base Alignment Quality (BAQ) adjustment (flag -baq 2). The folded SFS was then generated with ANGSD's companion program realSFS (flag -fold 1) with 200 bootstrap replicates and used to calculate heterozygosity as a percentage of heterozygous sites in R v.4.1.2 (R Core Team. 2015).

Genome Sequencing and Assembly
Sequencing on the PacBio Sequel II generated 196.7 Gbp of long-read data with a mean subread length of 9.79 kbp, yielding a coverage of approximately 79-fold. For the 10X Genomics Chromium library, we received 101.6 Gbp of raw sequencing data or 677 million short reads (41-fold).
The initial supernova run to assemble the Chromium data resulted in an assembly with a total length of only 1.9 Gbp and a scaffold N50 of 47.40 kbp. Therefore, we opted for a PacBio-based de novo assembly and used the Chromium data only for polishing. The final scaffolded and gap-closed chromosome-scale assembly (TBG_Okapi_asm_v1) has a total length of 2 387 734 269 bp in 3653 scaffolds and 3728 contigs, resulting in a contig N50 of over 61 Mbp and a scaffold N50 of more than 102 Mbp (Table 1A). The 28 largest scaffolds (>3.9 Mbp), which is five or six more than the expected haploid number of chromosomes for the okapi (2n = 44-46) (Ulbrich and Schmitt 1969;Petit et al. 1994) make up 98.0% of the total assembly length ( Figure 1B). The remaining contigs are smaller than 500 kbp. The five or six scaffolds that could not be anchored into the expected 44-46 chromosome-scale scaffolds lack enough evidence in the Hi-C contact map to be placed properly either by the algorithm of the HiRise pipeline or by manual curation. One reason for this could be the use of previously published Hi-C data from a different individual, which could exhibit a different karyotype than the individual used for the new assembly.

Assembly Completeness and Quality Assessment
GenomeScope estimated the genome size of the okapi at 2 526 089 448 bp, which is 140 Mbp larger than the total assembly length of TBG_Okapi_asm_v1 but 360 Mbp smaller than ASM166083v1_HiC. The coverage-based approach used in backmap estimated the haploid genome size at 3.07 Gbp, nearly 500 Mbp larger than the k-mer based estimate. Despite the discrepancy in genome size estimates, Merqury estimated an assembly completeness for TBG_Okapi_asm_ v1 of 91.89% and a QV of 32.79 based on k-mers, corresponding to a base error rate of 0.05%.
The gene set completeness analyses in BUSCO also suggest high completeness of the assembly identifying 94.7% of complete BUSCO genes in the Mammalia and 94.3% in the Cetartiodactyla dataset, the highest BUSCO scores of the three available assemblies ( Figure 1C,D). Furthermore, long and short reads map to the assembly with a mapping rate of 95.7% and 95.5%, respectively, and no contamination is evident in the BlobPlot (Supplementary Figure S1).

Genome-Wide Heterozygosity
The genome-wide heterozygosity values for the chromosomescale assemblies from ASM166083v1_HiC and the new assembly TBG_Okapi_asm_v1 were estimated at 0.120% (sd 9.24 × 10 -7 ) and 0.173% (sd 1.22 × 10 -6 ), respectively. These new estimates are in the same range as a previous estimate (0.132%) (Brüniche-Olsen et al. 2018) derived from the same short-read data as ASM166083v1_HiC. Even though these genomes are derived from captive individuals, the current levels of heterozygosity are likely to be representative for wild okapi in that the fully resolved captive okapi pedigree suggests no recent inbreeding. For example, Moyo, the individual sampled for TBG_Okapi_asm_v1, is the offspring of a female founder individual and an unrelated 1.5-generation male.
In comparison to other species, the heterozygosity of the two okapi individuals are around three to four times higher than the heterozygosity of wild individuals of the northern giraffe Giraffa camelopardalis (0.03-0.045%), which have a census population of approximately 5000 individuals (Coimbra et al. 2021. However, these estimates were derived using a different BAQ adjustment (-baq 1) in ANGSD, which is known to underestimate the heterozygosity by approximately three to four times (Prasad et al. 2022). Estimates from Hu et al. (Hu et al. 2020) place the heterozygosity of the okapi in the same range as other endangered species such as the Sumatran Rhinoceros Dicerorhinus sumatrensis (0.130%), the Giant Panda Ailuropoda melanoleuca (0.132%), and the Western lowland gorilla Gorilla gorilla gorilla (0. 144%). Yet, estimates of heterozygosity vary greatly depending on the methods used; hence, comparisons between studies must be evaluated with caution. Furthermore, heterozygosity alone may be an inaccurate representation of a species' level of threat of extinction from genetic depletion (Teixeira and Huber 2021), thus requiring complementary assessments of mutational load, inbreeding, and population sizes.

Repeat Annotation
Repeat annotation of TBG_Okapi_asm_v1 identified a repeat content of 43.53% or 1.04 Gbp of the assembly (Table 1B).

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Journal of Heredity, 2022, Vol. 113, No. 5 The most abundant retroelements are Long Interspersed Nuclear Elements (LINEs), which account for 31.09% of the assembly, whereas Long Terminal Repeats (LTR) elements, Short Interspersed Nuclear Elements (SINEs), and DNA transposons each only account for < 5%. The most common LINEs in TBG_Okapi_asm_v1 are RTE/Bov-B (15.27%) and L1/LINE1 (13.70%). Repeat statistics calculated for the two previously published assemblies show no major differences in repeat content compared to TBG_Okapi_asm_ v1, except of an approximately 2% lower repeat content in ASM166083v1_HiC likely attributed to the more fragmented assembly (Supplementary Table S1).

Gene Annotation
The homology-based gene prediction identified 23 412 genes in the TBG_Okapi_asm_v1 assembly with a median gene length of 7408 bp resulting in BUSCO scores of 88.8% and 90.1% for the Cetartiodactyla and Mammalia datasets, respectively ( Figure 1C,D). Of all 55 344 predicted proteins, 54 708 (98.85%) were functionally annotated by InterProScan, and in 41 684 proteins (75.31%) at least one GO term was identified. Furthermore, 53 839 proteins (97.27%) were assigned to the Swiss-Prot database.

Genome Synteny With Other Ruminants
Synteny analyses revealed high synteny between the two okapi assemblies (Figure 2A) with a few inversions and five additional scaffolds in TBG_Okapi_asm_v1. Manual curation of the Hi-C-based scaffolding yielded no evidence for the placements of the five additional scaffolds or the correct orientation of contigs responsible for the inversions in the synteny plot. As expected from cytological studies, the comparison between okapi and the Masai giraffe genome ( Figure  2B) revealed major rearrangements. In fact, not a single okapi scaffold could be found in the giraffe without being split into multiple parts or combined into the 15 much larger giraffe chromosomes. These chromosomal rearrangements, especially in the giraffe, are caused by Robertsonian-centric fusions of acrocentric chromosomes (Huang et al. 2008;Cernohorska et al. 2013;Agaba et al. 2016;Liu et al. 2021).
In contrast, the synteny between okapi and cattle or sheep was much higher ( Figure 2C,D), despite these species being more distantly related to the okapi than the giraffe. Many okapi scaffolds showed perfect synteny with scaffolds of the non-giraffid ruminants, indicating slow karyological evolution/changes.

PSMC
The estimation of effective population size (Ne) over time with PSMC revealed different trajectories for the two okapi genome assemblies TBG_okapi_asm_v1 and ASM166083v1_ HiC. For recent times (10-250 kya), both assemblies show congruent demographic patterns, with a maximum Ne around 40 kya. However, they diverge drastically further in the past (250 kya-2 Mya) reaching a 3-fold difference in Ne around 1.5 Mya (Figure 3). Differences in Ne estimates for such ancient time spans should be interpreted with caution, as they may reflect an artifact of the method. Further, both datasets are based on different library preparation methods (standard Illumina vs. 10× Genomics' Chromium), and to our knowledge, the effects of these methods on an analysis such as PSMC have not been tested. Yet, bootstrap analyses showed clear support for each of the two PSMC trajectories (Supplementary Figure S2). The third assembly (Okapi_RGD) could not be analyzed in this study, due to major differences in insert sizes in the available datasets, resulting in a much lower coverage after applying the same filtering of the BAM file as for the other datasets. However, Okapi_RGD has been previously analyzed with PSMC and showed a trajectory more similar to TBG_okapi_asm_v1 than ASM166083v1_HiC but with much more variability in the bootstrap replicates (Chen et al. 2019).

Conclusion
High-quality genome assemblies are an important resource for a variety of studies, especially for comparative genomics and conservation/ population genomics analyses. Currently, references are still missing for most non-model organisms, making the high-quality chromosome-scale genome assembly of the okapi presented here an important contribution to the field of evolutionary genomics. This is the first long-readbased assembly for this species, with the highest contiguity and completeness of BUSCO genes among the available okapi genome assemblies. Long-read-based assemblies are more likely to span complex repeat regions, such as telomeric or centromeric regions, which are usually collapsed in shortread-based assemblies, allowing for more in-depth analyses of structural variation (Gordon et al. 2016;Korlach et al. 2017;Paris et al. 2020).

Supplementary Material
Supplementary material can be found at Journal of Heredity online.

Funding
The present study was funded by the Centre for Translational Biodiversity Genomics (LOEWE-TBG) through the program "LOEWE -Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz" of Hesse's Ministry of Higher Education, Research, and the Arts.


s2fieldsofstudy: Biology Medicine

text:
Immunotherapy: TIMING OF DONOR SELECTION ON CD45RA-MEMORY T CELLS AS ADOPTIVE CELL THERAPY FOR COVID-19

Background & Aim: The COVID-19 pandemic has resulted in significant morbidity and mortality worldwide. The vaccines had dramatically decreased infection rates, number of deaths, and hospitalizations, but they are not 100% effective and immunity is lost gradually over time. We have previously shown how we are able to detect, isolate and produce at clinical scale SARS-CoV-2-specific T cells within CD45RA-memory T cells from COVID-19 convalescent donors. In a phase I clinical trial we have proved that treatment with these cells of hospitalized patients with moderate/severe COVID-19 is safe and feasible. Understanding the durability and the level of cellular immunity within the CD45RA- memory T cells and how changes with immunization are critical for the development of a biobank of living drugs to treat future COVID-19 patients. We performed a longitudinal exploratory analysis of the SARS-CoV-2 specific humoral and cellular immunity within the memory CD45RA- T cells in naive and previously infected individuals at different time points after two doses of BNT162b2 BioNTech/Pfizer vaccine Methods, Results & Conclusion: We studied the cellular and humoral response of SARS-CoV-2 specific memory T cells from recovered patients and controls at different time points: 2 weeks after recovering from COVID-19, 9 months after the infection/just before mRNA immunization, 10 and 65 days after full immunization. Detection of SARSCoV- 2- Specific Memory T Cells was performed by IFNg assay after exposure of cells to the M, N, and S SARS-CoV-2 peptides. Our data shows that memory T cell responses within the CD45RA- memory T cell subpopulation and most of the subsets tend to be higher in recovered individuals at all time points. The cellular response produced by control individuals to the S peptide is like the one from recovered patients at the same time point. Humoral responses were higher in recovered individuals after full immunization. Antibodies titer was not boosted after the late vaccine time point. An exploratory analysis of non-parametric Spearman’s rho correlation of humoral and cellular responses shows a positive correlation after infection with the 3 peptides and 65 days after immunization. In conclusion: We have analyzed the SARS-COV-2 specific T cells within the CD45RA- memory T cell subpopulation and the different subsets at different time points after (Figure Presented) (Figure Presented) infection and fully vaccinated. We claim that the best donors would be immunized individuals recovered from COVID-19 ideally in a time frame not higher than 6 months.

Background & Aim:
Background. Off-the-shelf, allogeneic CAR T cell therapies offer immediately accessible cell therapies to patients. High clinical response rates have been observed with  CAR T therapies, but opportunities for improvement remain. Strategies for investigating alternative cytotoxic effector cells that possess intrinsic tumoricidal activity, like  T cells, may improve depth and breadth of CAR T responses. Tumor targeting by allogeneic  CAR T therapy is complemented by innate and adaptive mechanisms. Studies have reported that tumor infiltrating  T cells are significantly correlated with patient survival and that V1 subset, primarily resident in peripheral tissues, can recognize and kill tumor cells through MHC-independent antigens. Aim. Here we describe extended preclinical evaluation, including gene-edited derivatives, of ADI-001, a non-gene edited allogeneic CAR V1  T cell therapy targeting CD20 designed for the treatment of B cell lymphomas.

Methods, Results & Conclusion:
Methods. Healthy donor-derived PBMCs were used to activate, expand, and genetically engineer V1  T cells to express a second-generation CAR targeting CD20 in an established manufacturing process. In vitro phenotype and antitumor activities of ADI-001 were assessed using flow cytometry and cell-based assays. Human tumor xenograft models in immunodeficient mice were used to evaluate in vivo efficacy. Additional characterization of gene-editing approaches to augment host versus graft influences were also compared to unmodified  T cell processes to evaluate relative susceptibility for host targeting. Results. PBMC-derived V1  T cells were successfully activated, expanded, and genetically engineered using established manufacturing processes. ADI-001 demonstrated a predominantly naïve-like T cell memory phenotype and expressed multiple chemokine and natural killer cell receptors. ADI-001 exhibited robust in vitro and in vivo tumor growth inhibition in multiple human lymphoma cell lines. No xenogeneic GvHD or other toxicities were induced by ADI-001. Lastly, comparison of gene-edited derivatives to ADI-001, including B2M KO with or without HLA-E overexpression, did not effectively limit elimination as compared to unmodified ADI-001. Conclusion. In summary, these findings demonstrate preclinical proof-of-concept for ADI-001 as an allogeneic CAR-T therapy. A phase 1 trial using ADI-001 to treat R/R B cell NHL patients is currently under investigation (NCT04735471).  Keywords: SARS-CoV-2 specific Memory T lymphocytes, Lymphopenia, COVID-19.

Background & Aim:
The COVID-19 pandemic has resulted in significant morbidity and mortality worldwide. The vaccines had dramatically decreased infection rates, number of deaths, and hospitalizations, but they are not 100% effective and immunity is lost gradually over time. We have previously shown how we are able to detect, isolate and produce at clinical scale SARS-CoV-2-specific T cells within CD45RA-memory T cells from COVID-19 convalescent donors. In a phase I clinical trial we have proved that treatment with these cells of hospitalized patients with moderate/severe COVID-19 is safe and feasible. Understanding the durability and the level of cellular immunity within the CD45RA-memory T cells and how changes with immunization are critical for the development of a biobank of living drugs to treat future COVID-19 patients. We performed a longitudinal exploratory analysis of the SARS-CoV-2 specific humoral and cellular immunity within the memory CD45RA-T cells in naive and previously infected individuals at different time points after two doses of BNT162b2 BioNTech/Pfizer vaccine Methods, Results & Conclusion: We studied the cellular and humoral response of SARS-CoV-2 specific memory T cells from recovered patients and controls at different time points: 2 weeks after recovering from COVID-19, 9 months after the infection/just before mRNA immunization,10 and 65 days after full immunization. Detection of SARS-CoV-2-Specific Memory T Cells was performed by IFNg assay after exposure of cells to the M, N, and S SARS-CoV-2 peptides. Our data shows that memory T cell responses within the CD45RA-memory T cell subpopulation and most of the subsets tend to be higher in recovered individuals at all time points. The cellular response produced by control individuals to the S peptide is like the one from recovered patients at the same time point. Humoral responses were higher in recovered individuals after full immunization. Antibodies titer was not boosted after the late vaccine time point. An exploratory analysis of non-parametric Spearman's rho correlation of humoral and cellular responses shows a positive correlation after infection with the 3 peptides and 65 days after immunization. In conclusion: We have analyzed the SARS-COV-2 specific T cells within the CD45RA-memory T cell subpopulation and the different subsets at different time points after Fig. 1 (abstract 508).
infection and fully vaccinated. We claim that the best donors would be immunized individuals recovered from COVID-19 ideally in a time frame not higher than 6 months. Keywords: T-Cell, Glioma, Immunotherapy.

Background & Aim:
High-grade gliomas (HGG) often present with poor survival rates due to their resistance to current treatment strategies. T cell-based immunotherapies have been shown to penetrate the BBB and precisely target proteins unique to malignant cells. These therapies have shown promising results against other malignancies. However, some patients with HGG progress too rapidly to allow autologous T cell manufacture, and there is evidence to suggest that T cell dysfunction is present due to treatment as well as disease related factors. An established bank of HGG antigen-specific T cells may circumvent these limitations. The use of "off the shelf" T cell therapies derived from allogeneic donors matched at one HLA allele or more have been successfully used for the treatment of viral infections. Therefore, we investigated the feasibility of developing an "off the shelf" PRAME-specific T cell therapy for HGG. Methods, Results & Conclusion: T cells derived from healthy donors were harvested from peripheral blood and expanded in vitro. Mature dendritic cells (DCs) were pulsed with overlapping peptides spanning the PRAME protein and were used to stimulate the harvested T cells. The specificity and cytotoxicity of these expanded cells were tested using IFN- ELISPOT and chromium release assays, respectively. T cells expanded a mean of 82.6-fold after three stimulations with DCs and were specific for PRAME (348.1 ± 74.5 SFC/1x10 5 cells) versus negative control (12.3 ± 4.4 SFC/1x10 5 cells) in IFNg ELISPOT assay. PRAME-specific T cell products HLA-matched with tumor targets at 4 of the tumor's HLA class I alleles specifically killed glioma cells (13.56% killing at E:T ratio of 40:1). In comparison, non-specific T cells derived from the same donor did not show specific tumor killing (2.79%). PRAME-specific T cells that only matched at 1 HLA allele showed lower killing (6.72%). However, PRAME-specific T cell products that were completely HLA-mismatched with the tumor did not elicit any anti-tumor activity (0% at 40:1 E:T) despite showing robust cytolytic activity against PRAME expressing autologous PHA blasts (20.42%). In summary, our preclinical results suggest that "off the shelf" PRAME-specific T cell therapies are a potentially viable treatment option for HGG. Future work will focus on testing the in vivo function in an in vivo orthotopic mouse model as well as clinically (IND27716).


text:
Sarco/endoplasmic reticulum calcium ATPase activity is unchanged despite increased myofilament calcium sensitivity in Zucker type 2 diabetic fatty rat heart

Systolic and diastolic dysfunction in diabetes have frequently been associated with abnormal calcium (Ca2+) regulation. However, there is emerging evidence that Ca2+ mishandling alone is insufficient to fully explain diabetic heart dysfunction, with focus shifting to the properties of the myofilament proteins. Our aim was to examine the effects of diabetes on myofilament Ca2+ sensitivity and Ca2+ handling in left ventricular tissues isolated from the same type 2 diabetic rat hearts. We measured the force-pCa relationship in skinned left ventricular cardiomyocytes isolated from 20-week-old type 2 diabetic and non-diabetic rats. Myofilament Ca2+ sensitivity was greater in the diabetic relative to non-diabetic cardiomyocytes, and this corresponded with lower phosphorylation of cardiac troponin I (cTnI) at ser23/24 in the diabetic left ventricular tissues. Protein expression of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), phosphorylation of phospholamban (PLB) at Ser16, and SERCA/PLB ratio were lower in the diabetic left ventricular tissues. However, the maximum SERCA Ca2+ uptake rate was not different between the diabetic and non-diabetic myocardium. Our data suggest that impaired contractility in the diabetic heart is not caused by SERCA Ca2+ mishandling. This study highlights the important role of the cardiac myofilament and provides new insight on the pathophysiology of diabetic heart dysfunction.

www.nature.com/scientificreports/ was increased in type 2 diabetic cp/cp rat hearts, but intracellular Ca 2+ concentration was not different at basal conditions 17 . Daniels et al. also recently found that cardiac function could be restored in diabetic rat trabeculae by kinase inhibition without any change in Ca 2+ flux 20 . These findings suggest that SERCA alone does not regulate the intracellular Ca 2+ state, and that altered intracellular Ca 2+ regulation is not the only factor causing contractile dysfunction in diabetic hearts. An alternative explanation for impaired contractility is that the Ca 2+ sensitivity of cardiac myofilaments is altered in the type 2 diabetic heart. The Ca 2+ sensitivity of the myofilament is affected by transient posttranslational modifications of myofilament proteins 21 , particularly cardiac troponin I (cTnI) 22,23 . In both type 1 and type 2 diabetic animal models, myofilament Ca 2+ sensitivity of cardiomyocytes has been reported as unaltered 6,24 , increased 25 or decreased 26 . The obese Zucker diabetic fatty (ZDF) rat is a type 2 diabetic model with well-established cardiac inotropic and lusitropic dysfunction 3,6,27 . Our group recently found that myofilament Ca 2+ sensitivity was increased in 20-week-old type 2 diabetic ZDF rat cardiomyocytes, and this was associated with reduced phosphorylation of cTnI at Ser23/24 28 . Interestingly, increased myofilament Ca 2+ sensitivity would be expected to enhance cardiac contractility by increasing the affinity of thick and thin filament interaction 29 . Therefore, it is unclear why contractile dysfunction still existed in these hearts. One possibility is that deranged SERCA activity reduces intracellular Ca 2+ concentrations. Reduced SERCA Ca 2+ uptake has been associated with reduced systole Ca 2+ transient amplitude and impaired cardiac contractility in type 2 diabetic mouse heart 11 . Reduced SERCA-mediated Ca 2+ uptake activity may decrease sarcoplasmic reticulum Ca 2+ content, hence less intracellular Ca 2+ would be available for the subsequent contractions, and this may explain systolic dysfunction in the diabetic heart.
As our group has recently found that myofilament Ca 2+ sensitivity is increased in type 2 diabetic heart 28 which is not normally associated with contractile dysfunction, we therefore hypothesised that SERCA expression and activity would be reduced in 20-week-old type 2 diabetic rat heart.

Discussion
The obese diabetic ZDF rat heart is characterised by depressed contractility but increased myofilament Ca 2+ sensitivity 28 . This implies that reduced Ca 2+ availability exists during systole in order to account for reduced force generation. In this context, we hypothesised that the activity of SERCA, the protein most responsible for the intracellular Ca 2+ flux, would be lower in the obese diabetic ZDF rat left ventricle. However, SERCA activity was not different between groups, despite the fact that the SERCA/PLB ratio was lower in the diabetic hearts. Similarly, the expression of NCX, which extrudes Ca 2+ from the cardiomyocyte, was not different between groups.  www.nature.com/scientificreports/ As previously shown 28 , we found that myofilament Ca 2+ sensitivity was greater, and that this augmentation was associated with lower phosphorylation of cTnI at Ser23/24 in the diabetic hearts compared to the non-diabetic hearts. Therefore, while left ventricular contractility is reduced in this diabetic heart model, our findings suggest this dysfunction is not caused by changes in SERCA activity. Increased myofilament Ca 2+ sensitivity in the obese diabetic ZDF rat cardiomyocyte suggests that less Ca 2+ would be required to elicit a given contractile force than in the non-diabetic cardiomyocytes. Numerous isolated heart experiments have shown that left ventricular force is reduced in this model 3,27 ; which when combined with increased Ca 2+ sensitivity, implies that the intracellular Ca 2+ concentration in diabetic cardiomyocytes is reduced; even in the absence of any difference in SERCA activity. Indeed, Belke et al. found that intracellular Ca 2+ concentration during systole and diastole was significantly lower in type 2 diabetic db/db mouse isolated myocytes 9 , which they attributed to decreased Ca 2+ handling by the sarcoplasmic reticulum. However, Zhang et al. argued that reduced expression of SERCA could explain prolonged action potentials and slower Ca 2+ transients, but not contractile deficits; which they attributed to remodelling of the myofilament, specifically a reduction in actin content 18 . Others have reported that reduced NCX activity impairs contractile function in type 1 diabetic rat models 30,31 . We did not quantify intracellular Ca 2+ transient amplitude in our experiments, but found no evidence to suggest that deranged Ca 2+ handling by either SERCA or NCX was associated with poor contractility in our experiment. Nonetheless, the combination of decreased contractility of isolated hearts and increased myofilament Ca 2+ sensitivity support the idea that intracellular Ca 2+ concentration may have been lower in the diabetic myocardium.
Although the protein expression of SERCA and PLB have been used as indicators of SERCA activity, our data, and others 12,32 suggest that protein levels do not necessarily reflect the intrinsic SERCA activity. We found that SERCA/PLB ratio and phosphorylated PLB at Ser16 were lower in the diabetic hearts, without any difference in the maximum SERCA Ca 2+ uptake rate between groups. The discrepancy between SERCA expression and activity may be explained by the 'coupling' between SERCA and PLB. Several studies have shown that only a fraction of SERCA is functionally regulated by PLB in the heart 33,34 . Brittsan et al. found that SERCA Ca 2+ uptake rate was proportionately reduced with increasing PLB expression in transgenic mouse model, and only ~ 40% of SERCA was under the inhibitory regulation of PLB in murine hearts under basal conditions 34 . This suggests that the 'coupling' between SERCA and PLB may not be affected by diabetes in the obese diabetic ZDF rat myocardium  www.nature.com/scientificreports/ and this may explain why SERCA Ca 2+ uptake activity was unaltered despite a lower SERCA/PLB ratio in the diabetic left ventricular tissues. Our finding that the diabetic ZDF rat heart had greater myofilament Ca 2+ sensitivity and lower phosphorylation of cTnI at Ser23/24 28 reinforces the inverse association between myofilament Ca 2+ sensitivity and phosphorylation of cTnI at Ser23/24 described in heart disease 21,35 . Similarly, our data show that phosphorylation of PLB at Ser16 was lower in the diabetic rat left ventricular tissues. Phosphorylation of cTnI at Ser23/24 and PLB at Ser16 are commonly recognised as PKA-dependent phosphorylation sites mediated by the cyclic adenosine monophosphate (cAMP) pathway 21,36 . Bockus and Humphries found that inotropic and lusitropic responses were impaired in type 1 diabetic mice following acute activation of PKA 37 . Thaung et al. also found that the expression of G s protein, which increases cAMP level during β-adrenergic stimulation, was reduced by 28% in this type 2 diabetic ZDF rat model 27 . Therefore, our data suggest that cAMP/PKA signalling may be impaired in the diabetic heart. Interestingly, Daniels et al. found that contractility and relaxation were restored in diabetic rat trabeculae without any change in sarcoplasmic reticulum Ca 2+ flux when Ca 2+ /calmodulin dependent kinase II (CaMKII) was inhibited 20 . Unfortunately, the authors did not determine whether this effect was associated with alterations in the phosphorylation of cTnI or other myofilament proteins known to alter Ca 2+ sensitivity.
Though not the focus of our study, the functional consequences of increased myofilament Ca 2+ sensitivity may be involved in diabetic diastolic dysfunction. Increased baseline systole 38 , prolonged systolic duration 39 and slower diastolic relaxation 13 have been identified as early manifestations of diabetes, which precede cardiovascular disease. The greater myofilament Ca 2+ sensitivity in the diabetic rat hearts in this study may suggest stronger binding of Ca 2+ ions to cardiac troponin C (cTnC), which would delay or reduce the rate of Ca 2+ dissociation from cTnC 40,41 . This increased affinity of Ca 2+ for the troponin complex would prolong thick and thin filament  www.nature.com/scientificreports/ interactions, thereby 'delaying' the onset of relaxation, leading to incomplete cardiac relaxation as often described in the diabetic heart 27 . Our data may indicate that diabetes impacts the force generating components of the myofilament itself. It is possible that diabetes reduced cardiomyocyte force generation by interfering with the Ca 2+ -myofilament interaction. We found that skinned diabetic cardiomyocytes tended to produce less maximal force (~ 25%, P = 0.06) when bathed in supra-physiological Ca 2+ (pCa 4.5). In such conditions, it is assumed that all thick and thin filament cross-bridges are activated, and any difference in force is explained by differences in the quantity of force producing cross-bridges per unit area. Jweied et al. similarly found that maximal force was 29% lower in diabetic human skinned cardiomyocytes (P = 0.08 vs. non-diabetic controls) 42 and van den Brom et al. found that maximal force was 68% lower in 14-week-old type 2 diabetic ZDF rats with impaired cardiac function 6 . Zhang et al. found that the area of the actin filament was 8% lower in streptozotocin-induced diabetic rat myofilaments, and that the sarcomeric organization of actin was disorganized compared to non-diabetic myofilaments 18 . A reduction in the content or quality of force generating proteins in the diabetic left ventricular cardiomyocyte would explain the consistent finding that maximal force is reduced in isolated heart experiments in this model 3,6,27 . It is also possible that the increase in myofilament Ca 2+ sensitivity would compensate for this loss of generated force by eliciting greater contractile activation at lower intracellular Ca 2+ concentrations.
Previous studies by our group have consistently shown that type 2 diabetic ZDF rats have impaired cardiac contractility 20,27 and coincide with increased myofilament Ca 2+ sensitivity 28 . SERCA is the primary Ca 2+ pump in cardiomyocytes; however, the lack of change in SERCA activity may indicate that an alternative Ca 2+ transporter is altered by diabetes. Several studies have suggested that NCX activity is increased in diabetic cardiomyocytes 8,11 , and may 'compete' with SERCA to remove Ca 2+ from the cytosol. We did not measure NCX activity in the present study. However, NCX expression was not different between the diabetic and non-diabetic myocardium, which may indicate a normal Ca 2+ efflux under basal condition. Similarly, Belke et al. found that NCX activity was not different between type 2 diabetic and non-diabetic hearts 9 . A more exhaustive comparison of the various Ca 2+ ion channels (i.e. L-type, NCX, ryanodine receptors, SERCA), and the dynamic interactions between Ca 2+ and the myofilaments may be necessary to better understand the mechanism of diabetic heart dysfunction.
Our data show that the type 2 diabetic left ventricular cardiomyocytes have greater myofilament Ca 2+ sensitivity and lower phosphorylation of cTnI at Ser23/24. SERCA/PLB ratio was reduced, however, SERCA Ca 2+ uptake activity was unaltered in the diabetic hearts, suggesting that impaired contractility may be associated with changes in intracellular Ca 2+ concentration independent of SERCA. These findings suggest an important role for myofilaments and generate new questions regarding the pathophysiological mechanism in diabetic heart dysfunction.

Methods
Animals. Experiments were conducted on 20-week-old male type 2 diabetic Zucker Diabetic Fatty (ZDF fa/fa ; n = 5) rats and their non-diabetic (homozygous ZDF +/+ or heterozygous ZDF +/-; n = 5) littermates. Animals were obtained from an in-house colony, with the original breeding pairs obtained from Charles River Laboratories (Wilmington, MA, USA). Animals were housed in groups of 2 per cage with a standard 12-h light-dark cycle (0700 -1900) and stable temperature of 21 ± 1 °C. Animals were fed with rat chow Purina 5008 (LABDIET) and had access to food and water ad libitum.
At 20 weeks of age, the animals were weighed and deeply anaesthetised with 60 mg/kg sodium pentobarbital via intraperitoneal injection. When the pedal withdrawal reflex was absent, the chest was cut open, the heart was quickly excised and rinsed in ice-cold Krebs-Henseleit buffer. The left ventricle was cut into several pieces, flash frozen in liquid nitrogen and stored at -80 °C. A thorax blood plasma glucose test was performed using a glucometer and test strips (ACCU-CHEK Performa, Roche) to confirm diabetic status.
Cardiomyocyte force-pCa measurements. Force measurements were performed on isolated skinned cardiomyocyte preparations at 15 °C as described by Diffee and Nagle 43 . The left ventricular tissues from the different groups were blinded and randomised to eliminate bias. A unique identification number was assigned to the left ventricular tissue of each animal and kept until all the measurements were completed. Briefly, left ventricular samples were defrosted in relaxing solution containing the following (mM): 100 KCl, 1.75 EGTA, 10 imidazole, 4 ATP-Mg, and 5 MgCl 2 ; pH 7.0. The left ventricular tissue was mechanically disrupted and incubated in relaxing solution supplemented with 1% Triton X-100 for 8 min to permeabilise the sarcolemmal membranes. The resulting isolated, skinned cardiomyocyte preparations were washed twice in relaxing solution. Thereafter, a skinned cardiomyocyte preparation (1-3 cells) was attached between a force transducer and a piezoelectric motor with silicon adhesive. Sarcomere length of the skinned cardiomyocyte preparation was adjusted to approximately 2.20 µm for isometric force measurements. The force was measured as a function of pCa (− log 10 [Ca 2+ ]) in solutions ranging from pCa 9.0 (relaxing solution) to pCa 4.5 (maximally activating solution).
Force was first measured in pCa 9.0 and 4.5 solutions and then in randomly ordered submaximal pCa solutions, followed by another measurement made in pCa 9.0 and 4.5 to assess any decline in cardiomyocyte performance. Measurements were continued in another three to four randomly ordered submaximal pCa solutions until a full force-pCa sigmoidal curve was obtained. This was followed by a last control measurement in pCa 9.0 and 4.5 to assess force rundown in the cardiomyocyte preparations. Skinned cardiomyocyte preparations without clear striations for sarcomere length measurement and that did not maintain more than 80% of maximal force at the end of the experimental protocol were discarded and data not used. For each activation, when steady-state force was reached, force was measured by abruptly slackened the skinned cardiomyocyte preparation by 20% of its initial length and transferred to pCa 9.0. Total force was calculated as the difference between steady-state force and the baseline force immediately after the skinned cardiomyocyte preparation was slacked. Active force www.nature.com/scientificreports/ was determined by subtracting passive force in pCa 9.0 from total force. Maximal force (force developed in pCa 4.5) and passive force (force developed in pCa 9.0) were normalised to the cross-sectional area (CSA) of the skinned cardiomyocyte preparation (CSA = 3.14 × (cell width/2) 2 ) . Active force at each pCa was expressed as a percentage of maximal force for that skinned cardiomyocyte preparation. Force-pCa relationships for each skinned cardiomyocyte preparation were fit to a modified Hill equation.
where F is steady state force, F max is the maximum isometric force at saturating Ca 2+ concentration, nH (Hill coefficient) is a measure of the steepness of the force-pCa relation which is an index of myofilament cooperative activation, pCa 50 is Ca 2+ concentration that produced 50% of maximal force which is used as an index of myofilament Ca 2+ sensitivity.
SERCA Ca 2+ uptake activity assay. Left ventricular tissue (~ 0.3 g) was mechanically disrupted in sarcoplasmic reticulum isolation buffer, which contained (mM): 290 sucrose, 10 imidazole, 3 NaN 3 , 0.5 PMSF, 0.5 DTT, 0.2 g/L trypsin inhibitor, and 0.2 g/L benzamide, pH 6.90. The tissue homogenate was centrifuged at 2300g for 15 min at 4 °C. The pellet was discarded and the supernatant was centrifuged at 50,000g for 3.5 h at 4 °C. After centrifugation, the supernatant was discarded and the pellet was re-suspended in 1 mL sarcoplasmic reticulum isolation buffer supplemented with 650 mM KCl, and incubated on ice for 45 min, followed by another centrifugation at 2300g for 10 min at 4 °C. The pellet was discarded and the supernatant was centrifuged at 50,000g for 4 h at 4 °C. The supernatant was discarded and the resulting pellet containing the isolated sarcoplasmic reticulum vesicles was re-suspended in isolation buffer, snap frozen in liquid nitrogen, and stored at − 80 °C for the SERCA Ca 2+ uptake assay. Ca 2+ uptake into the cardiac sarcoplasmic reticulum vesicles was measured according to the assay described by Beca et al. with some modifications 44 . The assay was performed at 30 °C in a 96-well plate with 100 µg of vesicles in a final volume of 100 µL of uptake buffer per well containing (mM): 100 KCl, 4 MgCl 2 , 20 HEPES, and 10 sodium oxalate, pH 7.0. The starting Ca 2+ concentration was adjusted to approximately 1.5 µM as assessed by the fluorescence ratio of fura-2. The reactions were initiated with a premix containing (mM): 1.5 Na 2 ATP, 1.5 creatine phosphate and 2 U/ml creatine phosphokinase. The fluorescence signals at 340 nm and 380 nm were recorded at 20-s intervals for 25 min with a multi-mode microplate reader (SpectraMax i3X, Molecular Devices). The rate of reaction for sarcoplasmic reticulum Ca 2+ uptake corrected for the amount of vesicle protein was calculated using the following formula: The data were plotted as rate of reaction against concentration of free Ca 2+ ([Ca 2+ ] free ), and the maximum Ca 2+ uptake rate was estimated from the plot by fitting a curve to the Michaelis-Menten equation.
The same uptake buffer was used for both diabetic and non-diabetic preparations, and the assay for both groups was run in parallel to minimise any potential variability in terms of the presence of heavy metals and Ca 2+ buffering capacity. In a separate experiment, thapsigargin, a potent SERCA inhibitor 45 completely inhibited Ca 2+ uptake in isolated sarcoplasmic reticulum preparation (see Supplementary Fig S2), confirming that the uptake of Ca 2+ was facilitated predominantly by SERCA. Sodium azide, a potent mitochondria inhibitor 46 did not affect Ca 2+ uptake, suggesting a lack of mitochondria-mediated Ca 2+ uptake in this assay (see Supplementary Fig S3).
Statistical analysis. Statistical analyses were performed using GraphPad Prism v.8.3.0. D' Agostino & Pearson test and Saphiro-Wilk test were used to check for a normal distribution. Student's unpaired t-tests were used to test for differences between non-diabetic and diabetic groups. Results are presented as means ± SEM, a value of P < 0.05 was considered statistically significant.